Mitochondria are responsible for generating over 90% of the cell's ATP, are implicated in aging (the site of oxidative damage in cells), and are found to play a key role in apoptosis and calcium homeostasis. For many of these mitochondria! functions, there is only a partial understanding of the components involved, with even less information on mechanism and regulation. Almost all biochemical studies on mitochondria so far are based on bulk assays in which millions of mitochondria are isolated and chemical analyzed. Yet mitochondria are pleomorphic organelles with structural variations depending on the cell type, cell-cycle stage, intracellular metabolic state, and even its precise spatial position inside the cell. Within a given cell, therefore, there can be a high degree of variability between individual mitochondria in terms of morphology and membrane potential, and potentially also the biochemical composition. In diseases that trace themselves to mitochondrial malfunction, both normal and abnormal mitochondria are thought to co-exist in a given cell. Bulk assays that average over millions of mitochondria will necessarily mask their distinctness. Here we propose to develop a new method for the chemical analysis of single mitochondrion. There is a number of the advantages associated with our proposed method of single mitochondrial analysis: (1) Single-mitochondrion isolation and analysis from a cell is rapid, which is important for studying labile species in the mitochondrion, such as the reduced form of glutathione. (2) The ability to physically extract a mitochondrion minimizes potential issues of contamination, which are concerns in bulk mitochondrial preparations (e.g. with differential centrifugation) in which other subcelluar organelles (e.g. endoplasmic reticulum) can be present. (3) Single-mitochondrial analysis permits the study of the variation between mitochondria, a fact that is especially pertinent given the highly variable nature of mitochondria in a given cell (a situation termed heteroplasmy). (4) We can correlate directly biochemical information from a given mitochondrion with its morphology (e.g. size and shape), which is important in the context of apoptosis, because one morphological signature of apoptosis is mitochondrial fragmentation. (4) We can directly correlate the biochemical information from a given mitochondrion with its precise spatial location in the cell. A single cell has been observed (using microscopy) to contain different types of mitochondria; with our method we can study directly such variations to better understand the physiological origin of these differences. While our focus here is on the analysis of individual mitochondria, we believe our method can be applied equally well to the chemical analysis of other subcellular organelles and compartments. [unreadable] [unreadable] [unreadable]